BIOLOGICAL OVERVIEW

The stereotyped pattern of veins in the Drosophila wing (see FlyBase for an interactive map of the wing) is generated in response to local EGF signaling. Mutations in the rhomboid (rho) gene, which encodes a sevenpass membrane protein required to enhance signaling transmitted by the EGF receptor (Egfr), inhibit vein development and disrupt the vein pattern. By contrast, net mutations produce ectopic veins in intervein regions. The net gene encodes a basic HLH protein that probably acts as a transcriptional repressor. net and rho are expressed in mutually exclusive patterns during the development of the wing imaginal disc. Lack of net activity causes rho expression to expand, and vice versa. Furthermore, ectopic expression of either net or rho results in their mutual repression and thus suppresses vein formation or generates tube-like wings composed of vein-like tissue. Egfr signaling and net exert mutually antagonizing activities during the specification of vein versus intervein fate. While Egfr signaling represses net transcription, net exhibits a two-tiered control by repressing rho transcription and interfering with Egfr signaling downstream of Rho. These results further suggest that net is required to maintain intervein development by restricting Egfr signaling, which promotes vein development, to the Net-free vein regions of the wing disc (Brentrup, 2000).

The specification of vein versus intervein fate in the Drosophila wing disc crucially depends on the restriction of Rho expression to the primordial veins. Rho enhances Egfr signaling, which generates elevated levels of activated Map kinase (MAPK) essential for proper vein development and the suppression of intervein fate in vein cells. In the absence of Egfr signaling, either in clones mutant for the Drosophila Egfr or in wings of double rhomboid;vein (rho^ve;vn¹) flies, veins fail to develop and assume an intervein fate. A counterpart of rho is net, whose transcription is confined to intervein sectors of third instar wing discs. Absence of net activity in third instar wing discs results in derepression of rho in all intervein regions except sector C (between veins L3 and L4) and initiates the formation of ectopic veins. Conversely, in the absence of rho activity, net is ectopically expressed in vein primordia of wing discs, as well as in distal veins L3 to L5 of pupal wings where differentiation of veins is suppressed. Thus, while net is repressed in vein cells by high Egfr signaling dependent on Rho, Net protein suppresses vein fate in intervein cells by repressing rho transcription. Repression of net in veins by high levels of Rho-dependent Egfr signaling is crucial for vein development, since ectopic expression of net is able to repress rho transcription and suppress vein fate. However, Egfr signaling in the absence of Rho is sufficient to initiate normal vein development in the proximal and anterior portions of the wing. Consistent with this finding, repression of rho by ectopic Net prevents vein formation only in regions where it depends on Rho (Brentrup, 2000).

Ubiquitous expression of rho or activated Ras represses net transcription in the entire wing disc and promotes vein development throughout the wing. Such flies have tube-like wings composed mainly, if not exclusively, of vein cells. Since this phenotype is much stronger than that of net null mutants, it follows (1) that additional factors that repress rho must be present in intervein regions, and (2) that ectopic Rho is able to suppress intervein fate in regions where such a fate is independent of net expression, and hence that in these regions rho is able to repress intervein-promoting and vein-suppression genes different from net. The first conclusion is also supported by the notion that rho does not expand into the intervein region between L3 and L4 in net mutants. The simplest explanation for these observations is that rho expression is controlled by a set of separate silencers responding to different repressors in different, perhaps overlapping intervein regions and that Net acts as one of the repressors of rho (Brentrup, 2000).

Net is a member of the bHLH transcription factor family, which has been subdivided into three structurally and functionally distinct classes. Since Net contains a valine residue in position 13 of the basic region of its bHLH domain, one might surmise that it is a class A protein and acts as a transcriptional activator. This assumption is consistent with the fact that Net has no class C-type repressor domains like the orange domain or the C-terminal WRPW-motif, which associates with the co-repressor Groucho. However, Net includes a proline-rich domain in its N-terminal moiety that might function as a repressor domain as shown for non-bHLH transcriptional regulators such as Even-skipped, Female-specific 1 and RGM1. Thus, despite the similarity of the Net bHLH domain to that of class A bHLH proteins, Net may act as a transcriptional repressor. This supposition is supported by the observation that Net represses rho expression during wing development. Although the possiblility cannot be excluded that Net activates a gene whose product represses rho, Net is also able to repress vein-promoting genes in the presence of activator proteins activated by ectopic Rho through enhanced Egfr signaling in interveins. The simplest explanation of these results is that Net is a transcriptional repressor and thus exceptional among class A proteins (Brentrup, 2000).

Any attempt to understand the role of net in the patterning of wing veins must consider its function in the context of the complex process of vein versus intervein specification, which occurs in several tiers of intimately linked gene regulatory circuits. The first distinction between vein and intervein anlagen is made during the third larval instar when the wing pouch is subdivided along the anteroposterior axis into sectors by prepattern genes activating morphogenetic signals, such as Hedgehog and Decapentaplegic, whose threshold concentrations determine the sector boundaries. Longitudinal veins are first specified by the activity of vein-organizing genes that are activated by short range signals in narrow stripes along these boundaries, while intervein genes, like Notch (N) and net, are thought to be activated through cues of prepattern genes. In the second tier of regulation, Egfr signaling plays a decisive role. Accordingly, one of the first genes to be activated by vein-organizing genes in third instar wing discs is rho. During late third instar and early prepupal stages, the membrane proteins Rho and S enhance signaling of the ubiquitously expressed Egfr, activated by the neuregulin-like protein Vein, and possibly additional ligands, in longitudinal veins and adjacent provein cells. High Egfr signaling activates Delta (Dl) as well as rho (Brentrup, 2000 and references therein).

Because Rho is able to stimulate Egfr signaling in adjacent cells, rho and Delta (Dl) expression continue to expand. Net is required to prevent the expansion of rho (and Dl) expression beyond the proveins into the intervein regions. These results suggest that Net might act in this process as a repressor of rho by binding to its wing enhancer, which is deleted in the rho^ve allele. Conversely, high Egfr signaling stimulated by Rho is essential in proveins to repress net, since net transcription is no longer confined to intervein cells in rho^ve mutant wings (Brentrup, 2000).

Dl expression in proveins initiates the third tier in the regulation of wing vein patterning. At the same time, Rho-dependent Egfr signaling begins to downregulate EGFR mRNA in proveins, yet MAPK activity remains high up to the early P2 pupal stage when crossveins become visible, presumably because of the continued stimulation of Egfr signaling through Rho. During this third tier of regulation, Dl activates N signaling in the lateral provein cells flanking the narrow central stripes of vein cells. N expression, which has been activated in a pattern complementary to that of Dl in third instar wing discs, is now enhanced by a positive feedback loop of N signaling in lateral provein cells. In addition, since N signaling represses rho transcription through the proline-bHLH protein E(spl)mß, expression of rho is again confined to the narrow stripes of vein cells by the early P2 pupal stage. This regulatory loop of N signaling appears to be analogous to that operating during lateral inhibition in neurogenesis, by which a neural precursor cell, stochastically selected from a group of proneural cells, prevents its neighbors from adopting a neural fate. However, in contrast to lateral inhibition, vein cells are not selected stochastically from provein cells, because longitudinal veins are straight, which suggests that expression of rho is always restricted to the central cells of the provein stripe. This is achieved by a fourth tier of vein fate regulation through Dpp signaling (Brentrup, 2000).

Expression of Dpp is activated by high Egfr signaling in provein cells at the same time as Dl expression. The effect of Dpp, however, is confined to the middle of the provein stripes by the secreted product of the short gastrulation (sog) gene, an antagonist of Dpp expressed in the adjacent intervein regions. If it is assumed that Dpp signaling inhibits expression of N, Notch signaling would be blocked only in the central cells of proveins, which are thus determined to become vein cells. Consistent with this last tier of regulation, veins in dpp minus clones are no longer straight, but follow an irregular path confined to the wider provein stripe. Dpp signaling maintains both its own expression and that of Rho in vein cells and determines their differentiation. Finally, the refinement of proveins to veins further depends on less well understood mutually inductive processes, in which predetermined vein cells of the dorsal wing surface signal to the underlying ventral cells to maintain vein cell identity (Brentrup, 2000 and references therein).

Vein cells are not only specified for their specific fate, but are also prevented from adopting the alternative intervein fate. This is achieved at all regulatory tiers by Rho-stimulated Egfr signaling, which represses, initially in proveins and later in veins, not only net but also blistered (bs). bs encodes a transcription factor, the Drosophila homolog of the mammalian serum response factor, which is instrumental in the specification of intervein fate. Like net and rho, activities of bs and rho repress each other and hence are mutually exclusive and expressed in complementary patterns of intervein and vein territories. However, whereas net and rho mutually depend on each other already from the onset of vein versus intervein development in third instar wing discs, bs and rho begin to restrict one another's expression only during prepupal development. Similar to net and rho, this mutual repression is not apparent in the proximal parts of the wing because of a redundancy inherent in this regulation. Although rho and net (or bs) are able to repress each other when expressed ectopically, no ectopic expression occurs in the absence of one of these genes in the proximal wing regions (Brentrup, 2000).

The activities of Rho and Net are not restricted to the mutual repression of their genes. This conclusion follows from the surprising observation that their ubiquitous co-expression in the developing wing generates a nearly wild-type wing. Since ubiquitous expression of Rho produces wings composed entirely of vein-like tissue, Net is able to repress vein-promoting genes in intervein regions, despite the presence of an activating signal stimulated by the ubiquitous Rho, while the situation is reversed in veins where Net is unable to repress vein-promoting genes in the presence of Net-independent Rho expression. Similarly, Rho-dependent signaling represses intervein-promoting genes, like bs, in veins, despite the presence of Net, but does not have this capability in interveins in the presence of Rho-independent Net expression. It follows that even in the absence of endogenous Net and Rho, vein and intervein primordia are in different states upon which ubiquitous Net and Rho can act to correctly determine their fates, which implies a considerable redundancy in the specification of vein versus intervein fate (Brentrup, 2000).

Because of the necessity for the concomitant suppression of the alternative fate when vein or intervein fates are specified, a system evolved in which Rho determines vein development by repressing net and later bs, which in turn specify intervein fate by repressing vein development in intervein regions. Such a balanced system is intrinsically labile unless it is stabilized through feedback loops. Multiple feedback loops operate at all tiers of vein fate regulation, and tiers are closely linked and overlap in time, which further enhances the stability of the system because it generates redundancy. It is proposed that the functions of Net and Bs are partially redundant because they both repress rho in intervein regions during overlapping, though not identical, developmental periods. Thus, while Net represses rho in all intervein sectors of third instar wing discs except sector C, Bs begins to repress rho in these regions only in early prepupal wings. In view of this hypothesis, it might be less surprising that the wing phenotype of net null mutants is much weaker than that resulting from ubiquitous expression of rho, which also represses net completely, but converts almost the entire wing into vein material. It is assumed that the lack of Net function in net minus wing discs is partially compensated by the activation of bs, whose product represses rho in most of the intervein regions during the prepupal and pupal stage. This assumption is consistent with the observations and with the earlier finding that bs null mutants exhibit a wing phenotype very similar to that resulting from ubiquitous expression of rho in the developing wing. The rho^ve -like phenotype obtained after ubiquitous expression of Net during wing development is largely explained by the ability of Net to repress rho. The partial redundancy of net and bs functions in wing discs is supported by experiments in which ubiquitous expression of Net is still able to suppress the strong ectopic vein formation phenotype of bs mutants (the phenotype is indistinguishable from that produced in a net¹ mutant background). In addition, bs expression is reduced in distal portions of net¹ wing discs and hence appears to depend partially on Net, a finding that is consistent with the observation that LacZ expression of a bs enhancer trap line is ectopically activated and enhanced after ectopic expression of Net in bs wing discs (Brentrup, 2000).

Since net and plexus (px) mutant alleles interact genetically and exhibit indistinguishable genetic behaviors and wing phenotypes (Díaz-Benjumea, 1990), one might surmise that they are part of the same developmental pathway. Indeed, px, which encodes a ubiquitously expressed nuclear matrix protein, like Net, is required for the repression of rho (Matakatsu, 1999). The finding that net expression is normal in px¹ or px⁷² mutant wing discs is unable to support a role for Px in the regulation of net activity. However, although px ⁷² is the strongest known px allele, at present no px null alleles are available to subject this possibility to a conclusive test (Brentrup, 2000).

The partial redundancy of net and bs might have evolved in more advanced insects like Diptera by the acquisition of vein-suppressing genes that reduce the much larger number of wing veins characteristic of more primitive insects. It has been proposed that such cryptic paraveins are still detectable in extravein mutants of Drosophila that display a strong tendency to misexpress rho and form ectopic veins between and parallel to L1 to L6. According to this hypothesis, net may have evolved to suppress paraveins P2 and P6, while hairy acquired the ability to suppress paraveins P4 and P5. It is attractive to speculate that the paravein-suppressing function of Net may have evolved as an ability to repress rho in paraveins, but not in vein primordia where net is not expressed, and thus entailed (evolutionarily speaking) a general vein-suppressing function, which initially may have been shared with bs. While bs later lost its early vein-suppressing function, its prepupal vein-suppressing function and pupal intervein differentiation function have been retained, which results in a temporal overlap of rho repression by Net and Bs and thus led to a temporal redundancy of net and bs functions. It is interesting to note that the rho repressing functions of net and h as well as that of E(spl)mß all encode bHLH proteins, a property that is entirely consistent with a general hypothesis of how gene networks evolve (Brentrup, 2000).

GENE STRUCTURE

cDNA clone length - 2185 bases

Bases in 5' UTR - 236

Exons - 2

Bases in 3' UTR - 816

PROTEIN STRUCTURE

Amino Acids - 365

Structural Domains

The net open reading frame encodes a polypeptide that includes a basic helix-loop-helix (bHLH) domain, which suggests that the Net protein is a DNA-binding transcription factor. The large superfamily of bHLH transcriptional regulators has been subdivided into three different classes. Members that belong to different classes bind to similar but distinct DNA recognition sites, thought to reflect characteristic differences in amino acids at specific positions of their bHLH domains. The Net protein is most closely related to bHLH proteins of class A, encoded by proneural genes like atonal and those of the Achaete-scute Complex. However, in contrast to class A proteins, which act as activators, Net contains a proline-rich domain in its N-terminal moiety (20% Pro between amino acids 100 and 164), which is characteristic of several transcriptional repressors. Diagnostic domains or motifs other than the bHLH and proline-rich domains have not been found (Brentrup, 2000).

date revised: 9 November 2000

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